The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17β-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 ± 5.3 fmol/3 days/106cells). The aromatase inhibitor letrozole (1 μM) and the pure estrogen receptor antagonist ICI 182,780 (10 μM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P<0.01) and almost 50% (P<0.001), respectively. Treatment with ICI 182,780 (10 μM) increased apoptosis by 228% (P<0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P<0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 μM) and letrozole (1 μM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P<0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.
A S Chagin, D Chrysis, M Takigawa, E M Ritzen, and L Sävendahl
J M Lean, J W M Chow, and T J Chambers
We have recently found that administration of oestradiol-17β (OE2) to rats stimulates trabecular bone formation. It is not known, however, whether oestrogen has a similar action on bone formation rate under physiological circumstances. Oestrogen is known to suppress bone resorption, and oestrogen-deficient states in the rat, as in humans, are associated with an increase in bone resorption that entrains an increase in bone formation. To see if the latter masks a relative reduction in bone formation, due to oestrogen deficiency, we measured bone formation very early after ovariectomy, before the resorption-induced increase in bone formation becomes established. To do this, rats were administered fluorochrome labels before and after ovariectomy, spaced at weekly intervals in the first, and 3-day intervals in the second experiment.
In both experiments there was a decrease in indices of bone formation in the labelling interval immediately following ovariectomy such that, using the shorter fluorochrome intervals, the mineral apposition rate fell to 69%, the double-labelled surface to 45%, and the bone formation rate to 36% of sham-ovariectomized levels. The reduction was not sustained in the subsequent label intervals, presumably masked by the increase in bone formation attributable to increased resorption. These results suggest that if bone formation is assessed before this resorption-entrained increase in bone formation occurs, oestrogen deficiency is associated with a reduction in dynamic indices of bone formation. Thus, these experiments suggest that oestrogen stimulates bone formation under physiological circumstances, and that the osteopaenia that follows oestrogen deficiency may be attributable not only to an increase in bone resorption, but also to a relative deficiency in bone formation.
Journal of Endocrinology (1994) 142, 119–125
Dimitrios Agas, Guilherme Gusmão Silva, Fulvio Laus, Andrea Marchegiani, Melania Capitani, Cecilia Vullo, Giuseppe Catone, Giovanna Lacava, Antonio Concetti, Luigi Marchetti, and Maria Giovanna Sabbieti
IFN-γ is a pleotropic cytokine produced in the bone microenvironment. Although IFN-γ is known to play a critical role on bone remodeling, its function is not fully elucidated. Consistently, outcomes on the effects of IFN-γ recombinant protein on bone loss are contradictory among reports. In our work we explored, for the first time, the role of IFN-γ encoding plasmid (pIFN-γ) in a mouse model of osteopenia induced by ovariectomy and in the sham-operated counterpart to estimate its effects in skeletal homeostasis. Ovariectomy produced a dramatic decrease of bone mineral density (BMD). pINF-γ injected mice showed a pathologic bone and bone marrow phenotype; the disrupted cortical and trabecular bone microarchitecture was accompanied by an increased release of pro-inflammatory cytokine by bone marrow cells. Moreover, mesenchymal stem cells’ (MSCs) commitment to osteoblast was found impaired, as evidenced by the decline of osterix-positive (Osx+) cells within the mid-diaphyseal area of femurs. For instance, a reduction and redistribution of CXCL12 cells have been found, in accordance with bone marrow morphological alterations. As similar effects were observed both in sham-operated and in ovariectomized mice, our studies proved that an increased IFN-γ synthesis in bone marrow might be sufficient to induce inflammatory and catabolic responses even in the absence of pathologic predisposing substrates. In addition, the obtained data might raise questions about pIFN-γ’s safety when it is used as vaccine adjuvant.
Idris Mohamed and James K Yeh
Long-term aromatase inhibitor use causes bone loss and increases fracture risk secondary to induced estrogen deficiency. We postulated that alfacalcidol (A; vitamin D3 analog) could help prevent the Letrozole (L)-induced mineral bone loss. Fifty intact 1-month-old female rats were randomly divided into basal group; age-matched control group (AMC); L group: oral administration of 2 mg/kg per day; A group: oral administration of 0.1 μg/kg per day; and group L+A for a period of 8 weeks. Eight-week administration of L resulted in a significant increase in body weight, bone length, bone area, bone formation, and bone resorption activities when compared with the AMC group. However, the bone mass and bone mineral density (BMD) were significantly lower than the AMC group. Serum levels of testosterone, LH, FSH, and IGF-1 were significantly higher and serum estrone and estradiol were lower along with a decrease in ovary+uterus horn weight, when compared with the AMC groups. None of those parameters were affected by A treatment, except suppression of bone resorption activities and increased trabecular bone mass and femoral BMD, when compared with the AMC group. Results of L+A combined intervention showed that bone length, bone area, and bone formation activities were higher than the AMC group, and the bone resorption activities were lower and BMD was significantly higher than that of the L group. This study demonstrates that the combined intervention of L and A not only enhances bone growth, but also increases bone density, and the effects of L and A are independent and additive.
T Yamashita, I Sekiya, N Kawaguchi, K Kashimada, A Nifuji, YI Nabeshima, and M Noda
Unloading induces bone loss as seen in experimental animals as well as in space flight or in bed-ridden conditions; however, the mechanisms involved in this phenomenon are not fully understood. Klotho mutant mice exhibit osteopetrosis in the metaphyseal regions indicating that the klotho gene product is involved in the regulation of bone metabolism. To examine whether the klotho gene product is involved in the unloading-induced bone loss, the response of the osteopetrotic cancellous bones in these mice was investigated. Sciatic nerve resection was conducted using klotho mutant (kl/kl) and control heterozygous mice (+/kl) and its effect on bone was examined by micro-computed tomography (microCT). As reported previously for wild-type mice (+/+), about 30% bone loss was induced in heterozygous mice (+/kl) by unloading due to neurectomy within 30 days of the surgery. By contrast, kl/kl mice were resistant against bone loss induced by unloading after neurectomy. Unloading due to neurectomy also induced a small but significant bone loss in the cortical bone of the mid-shaft of the femur in the heterozygous mice; no reduction in the cortical bone was observed in kl/kl mice. These results indicate that klotho mutant mice are resistant against bone loss induced by unloading due to neurectomy in both cortical and trabecular bone and indicate that klotho is one of the molecules involved in the loss of bone by unloading.
Joyce Emons, Bas E Dutilh, Eva Decker, Heide Pirzer, Carsten Sticht, Norbert Gretz, Gudrun Rappold, Ewan R Cameron, James C Neil, Gary S Stein, Andre J van Wijnen, Jan Maarten Wit, Janine N Post, and Marcel Karperien
In late puberty, estrogen decelerates bone growth by stimulating growth plate maturation. In this study, we analyzed the mechanism of estrogen action using two pubertal growth plate specimens of one girl at Tanner stage B2 and Tanner stage B3. Histological analysis showed that progression of puberty coincided with characteristic morphological changes: a decrease in total growth plate height (P=0.002), height of the individual zones (P<0.001), and an increase in intercolumnar space (P<0.001). Microarray analysis of the specimens identified 394 genes (72% upregulated and 28% downregulated) that changed with the progression of puberty. Overall changes in gene expression were small (average 1.38-fold upregulated and 1.36-fold downregulated genes). The 394 genes mapped to 13 significantly changing pathways (P<0.05) associated with growth plate maturation (e.g. extracellular matrix, cell cycle, and cell death). We next scanned the upstream promoter regions of the 394 genes for the presence of evolutionarily conserved binding sites for transcription factors implicated in growth plate maturation such as estrogen receptor (ER), androgen receptor, ELK1, STAT5B, cyclic AMP response element (CREB), and RUNX2. High-quality motif sites for RUNX2 (87 genes), ELK1 (43 genes), and STAT5B (31 genes), but not ER, were evolutionarily conserved, indicating their functional relevance across primates. Moreover, we show that some of these sites are direct target genes of these transcription factors as shown by ChIP assays.
Jonathan J Nicholls, Mary Jane Brassill, Graham R Williams, and J H Duncan Bassett
Euthyroid status is essential for normal skeletal development and the maintenance of adult bone structure and strength. Established thyrotoxicosis has long been recognised as a cause of high bone turnover osteoporosis and fracture but more recent studies have suggested that subclinical hyperthyroidism and long-term suppressive doses of thyroxine (T4) may also result in decreased bone mineral density (BMD) and an increased risk of fragility fracture, particularly in postmenopausal women. Furthermore, large population studies of euthyroid individuals have demonstrated that a hypothalamic–pituitary–thyroid axis set point at the upper end of the normal reference range is associated with reduced BMD and increased fracture susceptibility. Despite these findings, the cellular and molecular mechanisms of thyroid hormone action in bone remain controversial and incompletely understood. In this review, we discuss the role of thyroid hormones in bone and the skeletal consequences of hyperthyroidism.
P. L. Selby and R. M. Francis
In the 40 years or so since Albright & Reifenstein (1948) noted the association of osteoporosis with hypogonadism in both females and males, much has been learnt about the actions of hormones on bone and the endocrine causes of osteoporosis. Whilst subsequent work has underlined the importance of sex steroids in the maintenance of skeletal integrity, it is apparent that osteoporosis is multifactorial in origin and that non-hormonal factors are also involved in the pathogenesis of bone loss.
Osteoporosis is characterized by a reduction in bone mass in the skeleton, associated with an increased risk of fracture. The bone mass at any age, and therefore the risk of fracture, is determined by three variables: the bone mass at maturity, the age at which bone loss commences and the rate at which it proceeds (Riggs & Melton, 1986). The peak bone mass at maturity is regulated by sex, race, other genetic
J H Tobias, A Gallagher, and T J Chambers
Although short-term administration of oestradiol-17β (OE2) stimulates cancellous bone formation in the rat, this is replaced by a tendency to suppression after prolonged treatment. Hence, in rats rendered osteopaenic by ovariectomy, OE2 administration fails either to induce a sustained increase in bone formation or to restore bone volume. A possible explanation for this failure is that OE2 also inhibits bone resorption, secondarily suppressing bone formation through coupling mechanisms. We therefore investigated whether the effects of OE2 treatment might be modified by intermittently stimulating bone resorption with retinoic acid (120mg/kg daily) for 4 out of every 20 days. We found, in a preliminary experiment using intact animals, that intermittent retinoic acid reduced cancellous bone volume, consistent with previously documented stimulation of bone resorption by retinoic acid. Rats were then rendered osteopaenic by ovariectomy, and given vehicle, retinoic acid and/or OE2. We found that animals treated with intermittent retinoic acid and OE2 showed a substantial increase in cancellous bone volume compared with ovariectomized animals treated with vehicle, retinoic acid alone or OE2 alone. Therefore, intermittent retinoic acid appears to cause a net increase in bone formation over resorption when given to ovariectomized animals in conjunction with OE2. We conclude that the effects of OE2 on cancellous bone are modified by intermittent treatment with retinoic acid, resulting in a substantial increase in bone volume.
Journal of Endocrinology (1994) 142, 61–67
T. J. Allain and A. M. McGregor
There is immense current interest in the effects of thyroid hormones on bone. This is largely due to concern that patients on thyroxine replacement therapy are at increased risk of developing osteoporosis; this concern follows a number of reports describing reduced bone mineral density in this group of patients. The issue is, however, uncertain and the purpose of this review is (i) to summarize what is known about the effects of thyroid hormones on bone at both an experimental and clinical level and (ii) to try to reach a greater understanding of the problem and its implications for patient management.
Bone remodelling requires the tightly coupled actions of osteoclasts and osteoblasts. A normal bone remodelling cycle takes approximately 200 days. Each cycle begins with activation of cells which become osteoclasts and start resorbing bone. This phase lasts for about 50 days and is terminated